Differences between reference intervals of blood counts of Brazilian adults with and without sickle cell trait according to laboratory tests from the National Health Survey

ABSTRACT Objective: To compare reference intervals (RI) of blood counts of Brazilian adults with and without sickle cell trait (SCT). Methods: Cross-sectional study, based on the National Health Survey, 2014-2015, composed of 8,952 individuals. The sample of patients with SCT was composed of 234 adults. The RIs of adults with and without SCT were compared in the study “Reference values for laboratory tests of blood count in the Brazilian adult population: National Health Survey”, by Rosenfeld et al. (2019). The parametric method and the Student's t test were used for comparison (p≤0.05). Results: There were statistically significant differences between RIs of adults with and without SCT as far as sex is concerned for hemoglobin, MCV, MCH, MCHC, white blood cells, absolute lymphocytes, mean platelet volume and RDW; At all ages, for white blood cells and RDW in men and for MCV, MCH, MCHC, mean platelet volume and RDW in women; Between 18 to 59 years, for MCH, MCV, MCHC, neutrophils, lymphocytes and platelets in men and in women for lymphocytes, red blood cells, white blood cells, neutrophils, eosinophils, monocytes and platelets; From 60 years old on, for hemoglobin and hematocrit in men and in women for hematocrit, white blood cells, neutrophils and platelets; In white, black and brown people for white blood cells, neutrophils and platelets (p<0.05). Conclusion: Brazilian adults with SCT had lower counts of hemoglobin, MCV, MCH, MCHC, white blood cells and higher RDW than without SCT. The results show the importance of genetic counseling and further research to support the proper management of this condition in Brazil.


INTRODUCTION
Sickle cell trait (SCT) is a condition described as usually asymptomatic 1,2 . Its etiology stems from the presence of sickle cell hemoglobin (HbS), the result of a mutation after the replacement of A by T in codon 6 of the hemoglobin beta chain. As a consequence, glutamic acid changes into the amino acid valine. Valine-type hemoglobin leads to the sickling of red blood cells when exposed to low oxygen threshold 1 . Affected individuals inherit the hemoglobin A (HbA) and HbS genes from their parents. Thus, they are heterozygous with an abnormal allele of the hemoglobin beta gene 1 .
The importance of screening 3 and genetic counseling in people with SCT is highlighted as a means to clarify people about the risk of having children with sickle cell disease (SCD), since it is inherited through an autosomal recessive gene from both parents 2 . In SCD, individuals have two copies of the beta globin variant and the primary hemoglobin present in their red blood cells (HbS), making them homozygous (HbSS) 4 . The morbidity and mortality and severity of SCD is associated with microvasculature occlusions with tissue ischemia, leading to pain crises; organic lesions with functional asplenia; cerebral vasculopathy; kidney, lung and heart failure 4,5 .
Although SCT is commonly asymptomatic, there is evidence of cases of sudden death 2 in affected people. Such cases happen because of complications from hypoxia, including sudden death 6 , hematuria, hyposthenuria, pulmonary embolism, splenic infarction 2,6 , urinary tract infection in women, end-stage kidney disease, polycystic kidney disease, heart attack, stroke, eye problems, and lower limb ulcers 4 .
SCT is widely distributed continental-wise 7 , being more frequent in people of African descent 1,2,8 . It is estimated that there are 300 million people with SCT around the world 1,3 , with the highest prevalence in Equatorial Africa, Arabia, India, Israel, Turkey, Greece and Italy, reaching a 50% prevalence in certain regions of these countries 7 . In Brazil, the prevalence varies between 2 and 8% 7 . Studies with data from the National Health Survey (PNS -Pesquisa Nacional de Saúde), between 2014 and 2015, identified a prevalence of 2.49% in adults 9 .
SCD, on the other hand, is among the genetic pathologies of greatest epidemiological importance in Brazil and in other nations 2 , considered as a public health problem by the World Health Organization (WHO) 3 . Worldwide, in 2010, SCD affected 305,800 newborns, and an increase is expected to 404,200 by 2050 10 . In Brazil, the prevalence ranges from 1.1 to 9.8% and from 0.8 to 60 per 100,000 live births in different regions of the country 2 .
Changes in hematological parameters have been identified in people with SCD 8,11 . In people with SCT, although hematological parameters are usually described as normal or without important laboratory alterations 4 , this condition can lead to vaso-occlusion due to rigid erythrocytes in the face of pathological processes that cause hypoxia, acidosis, dehydration, hyperosmolality or hypothermia 11 .
Differences in the hematological parameters of people with SCT were found in populations from other countries, which means that the reference intervals (RI) of blood counts may suffer geographic and ethnic influences 3,8 . However, studies on the hematological profile of patients with SCT in Brazil are scarce. This study steps forward by providing, in an unprecedented way, through PNS exams, the hematological parameters of adults with SCT and the differences found in parameters of adults without SCT in the country. Our findings may contribute to support the handling of SCT in Brazil.
Thus, this study aimed to compare the RI of blood counts of Brazilian adults with and without SCT.

Study Design
Cross-sectional study with data from laboratory tests of the PNS carried between 2014 and 2015.

Context and data source
The PNS is a national household-based survey carried out by the Brazilian Institute of Geography and Statistics (IBGE -Instituto Brasileiro de Geografia e Estatística), in partnership with the Ministry of Health 12 .
The PNS 2013 used probabilistic sampling by clusters in three stages, with stratification of the primary sampling units (UPA), being selected in the first, second and third stages, respectively: census tracts; 10 to 14 households in each UPA; and one resident over the age of 18. At all stages, the selection processes were carried out by simple random sampling (SRS). Records of 64,348 households were obtained, of which 60,202 were interviews with adults. Furthermore, the collection of exams was planned in a subsample of 25% of the census sectors, being collected, between 2014 and 2015, from 8,952 adults 12 .
Due to the complex sampling design of the PNS and the unequal selection probabilities, sample weights were used 12 . In order to used the PNS laboratory database, weights were calculated by post-stratification procedures by sex, age range, race/skin color and level of education according to major region, from the total sample of the research 12 .
Laboratory samples were collected at any time of the day and without fasting. For blood count collection and hemoglobin electrophoresis, tubes with ethylenediaminetetraacetic acid (EDTA) were used. The samples were analyzed using an automatic cell analyzer 12 . Hemoglobinopathies were searched by the HPLC method, and the results of individual tests were interpreted, providing normal, homozygous or heterozygous parameters for Hemoglobin S (HbS), Hemoglobin C (HbC), Hemoglobin D (HbD) and oth-https://doi.org/10.1590/1980-549720230003.supl.1 er hemoglobinopathies 9 . More methodological details are available in other publications 12,13 .
The PNS database and questionnaires used in this study are available at: www.pns.fiocruz.br.

Participants
Participants were adults aged 18 and over. The PNS database, composed of 8,952 individuals, was used. A total of 330 adults were excluded due to absence of test results, insufficient material, sample loss and hemolysis 9 . In this study, data from 234 adults with SCT were analyzed. Data analyzed from adults without SCT were extracted from the study by Rosenfeld et al. 14 .

Reference Intervals
Intervals of adults with sickle cell trait: The RI of blood count parameters (described below) of adults with SCT were estimated considering the lower limit (LL) as the mean -1.96 SD (standard deviation) and the upper limit (UL), the mean +1.96 SD (standard deviation).

Intervals of adults without sickle cell trait
For adults without SCT, the means and RI (LL and UL) of blood count parameters from the study by Rosenfeld et al. 14 were used.

Statistical analyses
The RIs were estimated by the parametric method. The sample was partitioned according to sex, age and race/ skin color. For each partition, the means, SD and RI linked to the LL and UL were calculated (mean ± 1.96 SD) according to sex, age group and race/skin color. As the PNS sample was large enough and close to normal distribution, the Student's t test was used to compare RIs of adults with and without SCT in the study by Rosenfeld et al. 14 . The significance level adopted was 5%.
Data analyses were performed using the Data Analysis and Statistical Software (Stata), version 14, with the set of commands in the survey module, which considers post-stratification weights.

Ethical Aspects
The PNS was approved by the National Research Ethics Committee of the National Health Council (Opinion No. 328,159). Adult´s participation was voluntary, and confidentiality of information was guaranteed 13 .

RESULTS
For the red series, there were statistically significant differences for some blood count parameters when comparing RIs of adults with and without SCT 14 (p≤0.05) (Table 1). Men and women with SCT had lower mean values of hemoglobin, VCM, MCH, MCHC (Table 1) when compared to those without SCT 14 , as one can see from the means and RIs of the study by Rosenfeld et al. 14 displayed in Table S1. For RDW, the means of men and women with SCT were higher than those without SCT 14 . Men with SCT still had lower hematocrit means than those without SCT 14 (Tables 1 and S1).
Regarding the white series, for both sexes, statistically significant differences were observed in the RIs of some parameters of the blood count of men and women with SCT compared to those without SCT 14 (p≤0.05) ( Table 2). The means of white blood cells, neutrophils, lymphocytes and platelet volume were lower in men and women with SCT than in those without SCT 14 . Women with SCT also had higher mean eosinophils and platelets values than those without SCT 14 , and men with SCT had lower mean platelets than those without SCT 14 (Tables 2 and S1).
With regard to age, for the red series, there were statistically significant differences in the RIs of men with SCT compared to those without SCT 14 (p≤0.05) (Table S2). Means were lower between 18-and 59-year-olds for MCH, MCV and MCHC, and from 60 years onwards for hemoglobin, hematocrit and MCH in men with SCT than in those without SCT 14 . RDW means were higher in men aged between 18 and 59 years old with SCT than in those without SCT 14 . The means of hemoglobin, MCV, MCH and MCHC in women aged between 18 and 59 years and 60 years or older were lower in those with SCT than in those without SCT 14 . Women with SCT aged 60 years or older had lower hematocrit means compared to those without SCT 14 . Higher mean red blood cells and RDW were found in women aged 18 to 59 years with SCT compared to those without SCT 14 (Tables S2 and S3).
For the white series, there were statistically significant differences in the RIs of adults with and without SCT 14 when stratified by age (p≤0.05) (Table S4). Mean white blood cell values were lower in men with SCT than in those without SCT 14 aged between 18 and 59 years and 60 and older. Lower means of neutrophils, lymphocytes, platelets and platelet volume were identified in men with SCT when compared to those without SCT 14 in the two age groups. For women with SCT, the mean platelet volumes were lower in both age groups than for those without SCT 14 and aged 60 and older, women with SCT had lower mean white blood cells, https://doi.org/10.1590/1980-549720230003.supl.1 neutrophils and platelets compared to those without SCT 14 . Also in women with SCT compared to those without SCT 14 , higher means of white blood cells, neutrophils, eosinophils, monocytes and platelets were found in the age group 18-59 years (p≤0.05) (Table S3 and S4).
There were differences when comparing the RIs of some blood count parameters between men and women with and without SCT 14 of white, brown and black skin for hemo-globin, white blood cells, neutrophils and platelets (p≤0.05) ( Table S5). The means were slightly lower for both sexes for hemoglobin in brown adults with SCT than in those without SCT 14 . In white, black and brown men with SCT, the mean white blood cells, neutrophils and platelets were lower than in those without SCT 14 . White women with SCT had lower means of white blood cells and neutrophils than those without SCT 14 , and black women had lower platelet means.  In black and brown women with SCT, higher mean values of white blood cells and neutrophils were found, while for white and brown women, mean values of platelets were higher compared to those without SCT 14 (Tables S5 and S6).

DISCUSSION
This study assessed for the first time, using laboratory PNS data, the differences in hematological RI of Brazilian adults with and without SCT 14 . The findings show lower counts for hemoglobin, MCV, MCH, MCHC, white blood cells, lymphocytes and mean platelet volume, and higher counts for RDW in adults with SCT compared to those without SCT 14 , for both sexes. When stratifying according to sex and age, differences were identified in some parameters of white and red series when comparing adults with and without SCT 14 . Furthermore, differences were found in the RI of white, black and brown adults with SCT for white blood cells, neutrophils and platelets, and also in the brown adults for hemoglobin, when compared to those without SCT 14 . Such findings show the relevance of knowing the hematological profile of people with SCT for an adequate management of this condition in the country.
Bearing in mind that hematological parameters of people with SCT usually do not show important laboratory alterations 4 , the differences found in the RI of some parameters of the blood count of Brazilian adults with and without SCT 14 point to the need for further studies to better understand the clinical implications in people with this condition.
In this study, the results found for the blood count RIs in adults with and without SCT 14 , according to sex, were consistent with a study carried out in Ghana, in which individuals with and without SCD 8 were compared. With regard to the red blood count series, in Brazilian adults of both sexes with SCT, the altered values of hemoglobin, MCHC and MCV indicate anemia [15][16][17][18] , microcytosis [17][18][19] and hypochromia 16,17 , unlike adults without SCT, whose reference values were unchanged 14 . Furthermore, low MCHC values in adults with SCT may be associated with iron deficiency anemia 20 or thalassemias 21 . Other findings in these people with SCT refer to hypochromic microcytic anemia with high RDW, which is also characteristic of lack of iron from nutritional deficit, deficiency in absorption due to gastrointestinal alterations and chronic loss of this micronutrient 22 , as evidenced by the UL of RDW.
This finding is alarming due to the possibility of malnutrition in people with SCT in Brazil, especially when taking into account that chronic hemolysis results in greater availability of iron, and its deficiency is unlikely in SCD 23,24 . This highlights the need to intensify actions in the field of food security in Brazil, with special attention to the most vulnerable groups 16 . This situation was aggravated by the COVID-19 pandemic, which put Brazil back on the hunger map 25 , and may have contributed to the worsening of this scenario in people with SCT and SCD. In compliance with the national and international pacts made by Brazil to re-duce iron deficiency anemia by 2030, this should be one of the priorities in public health 16 .
Blood count analyses by age followed similar patterns of analyses by sex, occurring in men and women with SCT of all ages, when compared to those without SCT 14 , alterations in the RI of MCV, MCH and MCHC. Possible explanations are the conditions of access health and food, as well as health assistance, once the clinical variability of SCD is influenced by social and economic factors 26 -which may also be happening for SCT. Other possible explanations for the lower values of MCV, MCH and MCHC observed in adults with SCT are the effects of anemia of chronic disease, higher risk of infections and the degree of hemolysis in which such parameters are reduced 24 .
When analyzing the white blood count series according to sex, with regard to white blood cells and neutrophils, what draws attention are the even lower mean values found in Brazilian adults with SCT compared to those without SCT. In the analyses by age, the presence of leukopenia 27 is noted by the LL in all age groups and in both sexes, and the presence of leukocytosis 27 in women aged between 18 and 59 years identified by the UL. The literature describes that the decline in leukocytes is related to aging as a result of immune response to the extent that exposure to pathogens occurs 28,29 . Leukopenia can also result from viral and bacterial infections and medication use, and it is interchangeable with neutropenia 30 , as one can see by the LL of neutrophil found in Brazilian men and even women with SCT, whose mean values were slightly higher than in those without SCT 14 . Managing these situations requires identification of cause and effective therapy 30 . This finding possibly denotes greater clinical fragility of Brazilian adults with SCT exposed to malnutrition and infections.
As for the higher white blood cell count in Brazilian women with SCT aged between 18 and 59 years, when compared to those without SCT 14 identified by the UL, similar results were found in Nigeria, but in the SCD. 24 It is also worth mentioning that, in people with SCD, leukocytosis can occur due to autosplenectomy resulting from occlusion of splenic vessels, increasing vulnerability to infections 24 . Leukocytosis in SCD indicates a poor prognosis, being a risk factor for early death, stroke and acute chest syndrome, while a reduction in neutrophil counts is associated with a good prognosis 24 . There is evidence to suggest that the phagocytic function of neutrophils may be reduced in individuals with SCD, implying that their phagocytic competence affects the clinical severity of the disease 24,31 . All explanations lack empirical and theoretical evidence, so they need to be further investigated in adults with SCT.
As for the significant differences found in platelets, when stratified by sex and age, the highest mean values in women with SCT aged between 18 and 59 years compared to those without SCT 14 may stem from thrombocytosis, attributed to hemolytic anemia and autosplenectomy associated with SCD 24,32 . It is well established in the literature that https://doi.org/10.1590/1980-549720230003.supl.1 thrombocytosis is associated with SCD and several types of anemia 24,32 . The lower mean levels of platelets in men with SCT compared to those without SCT 14 and aged between 18 and 59 years may result from alterations in components of hemostasis, including platelet function, procoagulant mechanisms, anticoagulants and the fibrinolytic system in SCD 33 . The hypercoagulable state present in people with HbS is related to qualitative changes in platelets 33 , which would possibly explain these findings.
The other hematological alterations found in adults with SCT compared to those without SCT 14 , in women aged between 18 and 59 years for eosinophils and monocytes, and hematocrit after 60 years old, as well as those found in men aged 18 to 59 years old for lymphocytes and hemoglobin, and hematocrit at 60 years or older, can be attributed to behavioral, genetic, nutritional, socioeconomic factors, as well as exposure to allergens, infections and parasitic loads 34 .
In the present study, there were substantial differences in the hematological RI of Brazilian adults with and without SCT 14 according to skin color. Anemia identified by lower hemoglobin levels in brown adults with SCT compared to those without SCT 14 deserves to be investigated, given that conditions associated with anemia differ by ethnicity 35 . Although racial differences in hematological parameters may be due to heredity and chronic diseases, socioeconomic factors differ according to ethnicity as well 35 . The literature reports the lack of studies that explore racial differences in hematological parameters 35 . Black populations are underrepresented in epidemiological studies on the subject 35 , and we also identified the lack of investigations in the brown population. This is an important point of discussion, given that the Brazilian population is characterized by miscegenation, different ethnicities and social/economic segments 14 , which has implications for the epidemiology of SCT and SCD in the country 36 .
This study had limitations inherent to cross-sectional studies, such as the impossibility of establishing a causal relationship; some findings in blood count parameters may reflect lifestyle and treatment. However, the hematological profile of Brazilian patients with SCT has not been studied extensively in recent years, and this is the first study with this purpose. Given the representative sample of the Brazilian population, the generalization of results is relatively safe for national estimates. It was not possible to identify people with SCD due to unavailability in the PNS database; laboratory alterations may lead to underdiagnosis of SCD in the country. Thus, our results must be interpreted with caution. Furthermore, there is controversy in the literature whether SCT is a benign state or an intermediate phenotype of the disease, and at the same time, we lack studies on this condition and its complications, which could be underestimated 37 . Evidence suggests that SCT may not be a completely benign state nor a disease, but a risk factor for adverse events resulting from the interaction between genetic and environmental influences 38 .
This study showed differences between the RI of Brazilian adults with and without SCT 14 . These results point out the importance of neonatal screening to identify SCT, genetic counseling and the need for research that characterizes the consequences and risks to health, to support the control and prevention of diseases. The detection of individuals with heterozygous hemoglobinopathies such as SCT is extremely relevant in terms of public health 39 for its adequate management in Brazil.